Clostridium difficile causes disease within the gastrointestinal tract, usually following an alteration of the intestinal microecology. The pathogenic role of toxigenic C. difficile in antibiotic-associated pseudo-membranous colitis in humans is well established. Disease symptoms vary with the source of the pathogen, antibiotic exposure, toxin production, and patient age.
C. difficile produces two toxins, designated A and B. The toxins are implicated in the etiology of the disease. Toxin A is primarily an enterotoxin with slight cytotoxic activity, whereas toxin B is a potent cytotoxin. Toxin A causes extensive damage to the gut mucosa, as well as accumulation of fluid in the intestinal tract. It is believed that the primary event in the mechanism of C. difficile infection involves the specific binding of toxin to receptors on the intestinal cell surface. C. difficile-associated intestinal disease has been reported in infants, and in adults inthe absence of antibiotic therapy. Moreover, C. difficile is one of the most common bacterial enteropathogens found in stool specimens in hospitals. The organism has been reported to be one of the most commonly detected bacterial pathogens of enteric disease. C. difficile causes pseudomembranous colitis in humans as a result of the elimination of the normal flora of the colon by antibiotic usage, and growth of this toxin-producing bacterium. The disease usually occurs in hospitalized patients where it causes a massive diarrhea with extensive inflammation of the colon. Mortality rates as high as 44 % have been reported for this disease. Treatment is based on a proper diagnosis which is accomplished by establishing the presence of the toxin and demonstrating the characteristic lesions in the colon.
One method for detecting pathogenic C. difficile involves the culture of human feces, which requires specialized facilities for a long period of incubation. This test suffers from interference by non-pathogenic C. difficile strains, namely strains not producing toxin. The test is costly, time-consuming, and can only be performed in larger, well-equipped hospitals or in private laboratories.
Specific antisera to toxin A have been used to detect toxin A by means of an enzyme-linked immunosorbent assay (ELISA). Lyerly, et al., J. Clin. Microbiol. 17, 72-78 (1983). Similarly, the resulting immobilized antibody agglutinates soluble toxin A. The agglutination of the large latex beads may be visualized, indicating the presence of toxin A in the sample.
Lyerly et al., J. Clin. Microbiol. 21, 12-14 (1985) disclose a monoclonal antibody, affinity-purified polyclonal antibody and monospecific antiserum against toxin A as possible immunodiagnostic reagents for C. difficile disease.
U.S. Pat. No. 4,863,852 describes a method for isolating, detecting and purifying C. difficile toxin A utilizing a biological receptor for toxin A comprising a reagent containing the terminal oligosaccharide sequence Gal.alpha.(1.fwdarw.3)Gal.beta.(1.fwdarw.4)GlcNAc. The carbohydrate does not exist on human cells. Maximal binding of the toxin A to the Gal.alpha.(1.fwdarw.3)Gal.beta.(1.fwdarw.4)GlcNAc receptor occurs at 4.degree. C. At 37.degree. C., binding is not detected.